Illumina MiSeq paired end sequencing; Human gut microbiota, COVID-19 patients with mild or severe symptoms.
Human gut microbiota, COVID-19 patients with mild or severe symptoms.
Each sample were collected with a rectal swabs according to manufacturer's instructions. Samples were treated with 500 μl of Lysis Buffer ATL (QIAGEN, Hilden, Germany) at 56°C for 10 min with 20 μl Proteinase K (Darmstadt, Germany) before DNA extraction.  Microbial DNA was extracted from 500 μl of sample using Nimbus automatic extractor (Seegene Technologies, Korea) according to the manufacturer's protocol. The V3–V4 hypervariable region of the bacterial 16S  rRNA gene was amplified from total DNA according to the Illumina 16S Metagenomic Sequencing Library Preparation instructions, as previously described in Diamanti-Picchianti A, et al (Int J Mol Med ). In summary, the V3-V4 amplicon was obtained by PCR with universal primers, followed by purification. A second PCR was then performed to barcode the libraries using the Illumina dual-index system. Following a second step of purification, libraries were diluted to 4nM and pooled.